Autofluorescence PTIR Imaging Without Labels or Coherent Artifacts
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Solution Overview
Problem
Conventional infrared spectroscopy and fluorescence microscopy techniques face challenges in achieving high sensitivity and spatial resolution without the need for external fluorescent labeling, and they suffer from coherent interference artifacts due to multiple reflections and scattering of probe light.
Innovation Solution
A method and apparatus utilizing autofluorescence-enhanced photothermal infrared spectroscopy (AF-PTIR) that excites autofluorescent emission in a sample using a modulated infrared beam, detects autofluorescent emission with an array-based detector, and constructs an output indicative of infrared absorption using two autofluorescent datasets, while employing a counterpropagating geometry to eliminate coherent artifacts.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional infrared spectroscopy is used, then chemical characterization is achieved, but spatial resolution is insufficient
Solution Approach 1:
The patent combines infrared spectroscopy with fluorescence microscopy to create a hybrid system that achieves both high spatial resolution and chemical analysis capability. The confocal microscope objective focuses infrared light to achieve micrometer-scale spatial resolution while simultaneously collecting fluorescence signals for chemical identification.
Solution Approach 2:
The patent uses fluorescence emission as an intermediary signal to enhance the detection of infrared absorption features. By detecting the fluorescence signal modulated by infrared absorption, the system achieves enhanced sensitivity and spatial resolution beyond conventional infrared spectroscopy.
2Measurement precision
If external fluorescent labeling is used to enhance signal, then sensitivity is improved, but sample complexity and potential artifacts increase
Solution Approach 1:
The patent utilizes the sample's own autofluorescence properties to enhance the infrared absorption signal without requiring external fluorescent labeling. The sample's intrinsic fluorescent molecules provide the necessary signal modulation, eliminating the need for additional reagents and reducing sample preparation complexity.
3Measurement precision
If probe light is used for infrared spectroscopy, then chemical information is obtained, but coherent interference artifacts occur due to multiple reflections
Solution Approach 1:
The patent converts the harmful coherent interference artifacts into beneficial information by detecting the fluorescence signal modulated by infrared absorption. The fluorescence detection mode inherently rejects coherent interference patterns, transforming them from problematic artifacts into irrelevant background noise that does not affect chemical analysis accuracy.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Achieves high sensitivity and spatial resolution in infrared imaging and spectroscopy without external fluorophores, reducing bleaching effects and eliminating coherent interference artifacts, thereby enhancing signal-to-noise ratio and improving chemical analysis.
Implementation Method 1
illuminating a first region of the sample with a modulated infrared beam... illuminating the sample with a beam of excitation radiation to excite autofluorescent emission
Implementation Method 2
The frequencies of infrared light, especially mid-infrared light (2.5-20 μm in wavelength) correspond to vibrational frequencies in molecular bonds. Thus, when a sample is illuminated by mid-IR light, it will absorb light at IR radiation frequencies corresponding to specific molecular vibration of chemical species in the sample.
Implementation Method 3
illuminating the sample with a beam of excitation radiation to excite autofluorescent emission in a second region
Data Source
AI summary
Methods and systems described herein detect autofluorescence of a sample. These methods and systems obviate the need for addition of fluorophores to samples to create IR absorption